Production methods employing recombinant technology use genetic expression systems. These systems generally consist of host cells encompassing a genetic system to be expressed, and expression vectors which introduce the genetic expression capabilities into the host cells. Under conditions allowing expression, a product, generally a protein, is made by the host cells.
Problems in commercial use of genetic expression systems arise because host cells have a variety of endogenous proteases, each with a specific action that may degrade the product. Degradation of product may also decrease the shelf lives of the bulk protein product and of the final dosage form of drugs.
Endogenous proteases degrade substrates in different ways. Aminopeptidases have broad substrate specificity, e.g., leucine aminopeptidase (Hanson and Frohne, 1976). However, when a proline residue is reached during degradation, such enzymes are unable to further degrade the peptide. Aminopeptidase P enzymes hydrolyse aminoacyl-proline bonds when proline is in the penultimate position from the amino terminus (X-Pro) of a polypeptide (Yoshimoto et al., 1988). After that action, proline aminopeptidase is capable of removing the exposed amino terminal proline residue.
Dipeptidyl peptidases have been found in many eukaryotic species (Kreil, 1990), but only in a few prokaryotic species (Lloyd et al., 1991; Fukusawa and Harada, 1981). These enzymes can remove N-terminal dipeptides including X-Pro dipeptides.
Tripeptidyl aminopeptidases are capable of degrading a peptide or polypeptide at its amino terminus by removing an amino acid triplet. Serine proteases from human, rat and pig tissues with tripeptidyl aminopeptidase activity have been characterized (McDonald et al., 1985, Balon et al., 1986), and a cDNA sequence has been reported (Tomkinson and Jonsson, 1991).
Various bacteria are known in the art to secrete proteases. For example, Bacillus PB92 produces a protease that degrades casein and a tripeptide substrate (z-Gly-Pro-citrulline-PNA). Roig et al., Appl. Biochem. Biotechnol. 55:95 (1995). A serine exopeptidease that cleaves Leu or Phe from tripeptide substrates has been characterized in Bacillus. Sharipova et al. Biotechnol. 94--Ferment. Physiol. pages 31-33 (1994). B. licheniformis produces a serine protease that is inhibited by PMSF. Pavlova et al. Mikrobiologiya 57:398 (1988). See also Balaban et al. Biokhimiya 59(9):1393 (1994). Lactobacillus helveticus produces a prolyl dipeptidyl aminopeptidase, a di/tripeptidase, and other dipeptidases. Nowokokski et al. Appl. Microbiol. Biotechnol. 39(2):204 (1993). Lactococcus lactis produces a tripeptidase with specificity for, inter alia, (Leu).sub.3 and Leu-Gly-Gly. EP 440 303 (Bosman et al.; publication date Aug. 7, 1991). Salmonella typhimurium produces a tripeptidase. Strauch et al. J. Bacteriol. 156:743 (1983).
Endoproteases can also cause rapid degradation of secreted proteins. Serine proteases are widespread throughout the prokaryotes as are metalloproteases. A wide variety of cleavage site specificities have been observed in various microbial species. Enzymes which cleave adjacent to positively charged, negatively charged, and aromatic amino acids have all been reported.
Proteases may be neutralized by various methods including by using inhibitors and by constructing improved strains with impaired proteases. The use of protease inhibitors to prevent the degradation of proteins during their purification is well established for proteins derived from yeast and higher eukaryotes. This approach has also been employed in the isolation and purification of proteins generated as inclusion bodies from E. coli. The general method involves lysing the protein source in the presence of broad spectrum protease inhibitors. Such inhibitors may include leupeptin, EDTA, phenylmethanesulfonylfluoride, or pepstatin.
The application of protease inhibitors in a system involving a living organism is more delicate. EDTA increases the fragility of many microorganisms and can cause cell lysis. Some inhibitors may be taken up by the organism. Such a process may lead to cell death or a disruption of cellular functions. Ideally, a protease inhibitor employed under these conditions should 1) be soluble in the fermentation media, 2) inhibit the target protease as selectively as possible, 3) not inhibit cell growth, and 4) be cost-effective.
The use of improved strains with impaired proteases also can prevent degradation of proteins during production. Improved strains carrying deletional mutations in multiple protease-encoding genes have been made in Bacillus strains (Sloma et al, 1992). International Application Number PCT/US92/01598 of Omnigene, Inc. describes a Bacillus cell containing a mutation in the residual protease III gene resulting in the inhibition of the production by the cell of proteolytically active RP-III. In that case, the inactivation of the major protease allowed detection of other minor proteases which were still present in quantities sufficient to cause degradation of secreted products.
International Application Number PCT/US92/05532 of Amgen Inc. entitled "Isolation and Characterization of a Novel Protease from Streptomyces lividans" describes a protease called "Protease X" of S. lividans, its DNA and amino acid sequence, antibodies raised against such protease and a strain of S. lividans deficient in such protease. Protease X has different DNA and amino acid sequences than the proteases described in this application and cleaves different substrates than those described in this application.
A specific recombinant genetic expression system designated CANGENUS.TM. has been used to ferment and produce a variety of protein products, for example, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), and erythropoietin (EPO) (see Canadian Patent Numbers 1,295,563; 1,295,566; and 1,295,567; and U.S. Pat. No. 5,200,327).
Although the CANGENUS.TM. system has been successful in producing exogenous products, some undesirable proteases produced by expression of endogenous genes deleteriously affect the quality, quantity or stability of exogenous products.
Thus, a need exists to impair the action of these Streptomyces proteases. Among strategies which can be employed to meet this need are the use of inhibitors to inhibit the effect of proteases during the production processes and the use of improved strains which lack such proteases or which have impaired proteases.
Isolation of the protease genes could also be useful in the design of vectors directing the expression and secretion of heterologous proteins from Streptomyces. The promoter and signal sequence of such proteases could be used to enhance and direct the export of heterologous proteins from Streptomyces. The proteases themselves could be usefully employed to remove specific amino acid sequences, peptides or polypeptides from a protein. Furthermore, it would be useful if the level of expression of such proteases could be enhanced through mutation, selection or genetic engineering.